Gamma interferon (IFN-.gamma.) is a cytokine produced by activated helper T cells, one of the most characteristic activities of which is the upregulation of MHC class IIgene expression. Class II genes (HLA-DR, -DP and -DQ) of the human major histocompatibility complex encode polymorphic cell surface glycoproteins that are expressed in specific cells of the immune system, such as B lymphocytes, macrophages, monocytes and activated T lymphocytes.
The class IIgene products are essential for cellular interactions involved in the immune response, including antigen presentation to helper T cells. It has been shown that, in addition to other biological functions, IFN-.gamma. plays a modulatory role in antigen presentation by increasing the level of class IIgene transcription in class II-positive cells as well as inducing its expression in the otherwise class II-negative cells, which include epithelial cells, fibroblasts, endothelial cells and astrocytes [Taylor et al., Virus Res. 15:1 (1990)].
Abnormal levels or aberrant expression of class II gene products have been implicated in the onset of several autoimmune diseases such as experimental autoimmune encephalomyelitis [Massa et al., Proc. Natl. Acad. Sci. USA 84:4219 (1987)]. Interestingly, a neutralizing antibody to IFN-.gamma. has been shown to abrogate the effects of autoimmune disease in the (NZB.times.NZW) F1 mouse model system of systemic lupus erythematosus [Jacob et al., J. Exp. Med. 166:798 (1987)].
Induction of class IIgene expression by IFN-.gamma. is regulated in part at the transcriptional level [Basta et al., Proc. Natl. Acad. Sci. USA 85:8618 (1988); Blanar et al., Proc. Nat). Acad. Sci. USA 85:4672 (1988)]. Functional studies have focused on identifying the cis-acting elements that are important for regulated class II gene expression.
Chimaeric DNA constructs containing 5' promoter deletions of the HLA-DR.alpha. gene linked to a reporter gene such as the bacterial chloramphenicol acetyltransferase (CAT) gene have been transiently transfected into several types of cells. From these studies, sequences that are important for IFN-.gamma.-induced expression in glioblastoma cells have been identified [Basta et al., J. Immunol. 138:1275 (1987); Basta et al., supra], as well as regions important for constitutive expression in lymphoblastoid cells and inducible expression in HeLa cells [Sherman et al., Proc. Natl. Acad. Sci. USA 84:4254 (1987); Tsang et al., Proc. Natl. Acad. Sci. USA 85:8598 (1988); Sherman et al., Mol. Cell. Biol. 9:50 (1989); Tsang et al., Mol. Cell. Biol. 10:711 (1990)].
Within the regions that have been identified as functionally important, highly-conserved sequences designated as X, Y, Z/W-boxes and an octamer (O) binding site have been noted [Miwa et al., Proc. Natl. Acad. Sci. USA 84:4939 (1987); Dorn et al., Proc. Natl. Acad. Sci. USA 84:6249 (1987); Sherman et al., Mol. Cell. Biol. 9:50 (1989); Tsang et al., Mol. Cell. Biol. 10:711 (1990)]. Mutational studies have suggested the functional importance of these elements.
For example, all four elements (X, Y, Z and O) are necessary for B cell specificity, whereas only the X, Y, and Z boxes are required for IFN-.gamma. inducibility. In addition, the Z box and sequences flanking the Z and X boxes appear to help in establishing low levels of expression in T cells and uninduced cells.
X-box binding is defective in cells from patients with the combined immunodeficiency syndrome, a hereditary disease characterized by the absence of class II expression in all tissues [Reith et al., Cell 53:897 (1988); Sherman et al., Mol. Cell. Biol. 9:50 (1989); Tsang et al., Mol. Cell. Biol. 10:711 (1990)].
The cloning of some of the DNA-binding proteins including those recognizing the X and Y boxes has been achieved, but their presence does not appear to correlate precisely with IFN-.gamma.-induced expression [Liou et al., Science 242:69 (1988); Celada et al., Mol. Cell. Biol. 9:3097 (1989); Reith et al., Proc. Natl. Acad. Sci. USA 86:4200 (1989)].
Because of its involvement in autoimmune disorders, there is impetus for the development of agents that can antagonize the effects of IFN-.gamma.. Yet, it has long been known that such interferon also has beneficial antiviral and antiproliferative activities that are useful in the treatment of neoplastic and viral diseases. For that reason, efforts are also being made to find agonists of IFN-.gamma..
The search for such agonists and antagonists would be facilitated by the development of a fast and effective in vitro screening system.